A major question in HIV vaccine research is the development of protective host immune responses. The overall goal of this proposal is to test the hypothesis that immunization with quasispecies vaccines will provide broader immune responses than immunization with clonal vaccine and that this broader immunity will translate into protection from quasispecies challenge within the same clade (B) and/or different clades. Several studies supported by this grant have demonstrated that combination immunization with multiple SIV or SHIV antigens using prime and boost regimens can protect against clonal virus challenge. However, it has not been possible to protect against more complex virus challenges using recombinant vaccines; in contrast, live-attenuated vaccines are very effective. We hypothesize that the failure of subunit vaccines to provide broad protection is due to immune responses that are too narrow in specificity or inadequately mature. We propose to model antigen presentation during progressive viral infection by sequential vaccination with viral Envelopes that represent natural shifts in sequence over time during disease progression. In Aim 1, we will develop clade B and Clade A immunogens and use these to immunize by DNA prime and subunit boost with a combination SHIV-based vaccine presenting the major structural and regulatory genes of SIV and a set of multiple HIV-1 Envelope variants derived from patients infected with clade B or with clad A. The quasispecies members will be cloned from a patient who9has been studied longitudinally and whose "early" and "late" passage dominant quasispecies viruses have been characterized (see Core B, Mullins). Clonal, sequential, and simultaneous presentation of SIV and HIV-1 Envelope variants as part of combination DNA prime and subunit boosts will be tested in mice (Aim 2) and in macaques (Aims 3 & 4) for the ability to induce cellular and humoral and cellular immunity. In Aim 3, protective against homologous viral challenge in macaques will be tested with complex SHIV isolate from clade B. The homologous challenge virus will be a SHIV "swarm" constructed from mixtures of 5 individual SHIV clade B primary isolate macrophage-tropic and T-tropic envelope genes from this patient. In Aim 4, macaques will be immunized with clade A and B vaccines, and reciprocal heterologous challenges will be done with clade B and A SHIV viruses. Finally, in Aim 5 we will examine in detail the fine specificity of the humoral immune responses induced by these quasispecies vaccines in vaccinated-protected and vaccinated-infected macaques in these vaccine experiments and related experiments in Projects 1 (HU), 3(Bosch) and 4 (Greenberg) and correlate these with breakthrough viruses analyzed in Core B (Mullins). We view this strategy as a potentially highly effective method for generating high titer, high avidity, broad spectrum immunity as a means of developing an effective, save vaccine against HIV-1 in humans.